Diagnostic device and method

ABSTRACT

A method of separating a cell-containing sample into a substantially cell-depleted portion, and a cell-containing portion comprising at least one of a stem cell, a lymphocyte, and a leukocyte comprises a step in which the sample is received in a vessel with at least one flexible wall. In another step, an additive and particles are added to the sample, wherein the additive substantially binds to the at least one of the stem cell, lymphocyte, and leukocyte, and the particles and wherein the particles substantially bind to the at least one of the stem cell, lymphocyte, and leukocyte, and the additive, thereby producing a cell-containing network. In a further step, the network is separated from the substantially cell-depleted portion by applying a magnetic force.

This application is a divisional application of U.S. patent applicationSer. No. 12/054,383 filed Mar. 24, 2008, now issued U.S. Pat. No.7,871,813, which is a divisional application of U.S. patent applicationSer. No. 10/371,837 filed Feb. 20, 2003, which is continuation-in-partof U.S. patent application Ser. No. 09/949,314, which was filed Sep. 7,2001, now issued U.S. Pat. No. 6,821,790, which is acontinuation-in-part of U.S. patent application Ser. No. 09/514,686filed Feb. 28, 2000, now issued U.S. Pat. No. 6,291,249, and acontinuation-in-part of U.S. patent application Ser. No. 09/261,068filed Mar. 2, 1999, all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The field of the invention is clinical diagnostics and biotechnology.

BACKGROUND OF THE INVENTION

In vitro diagnostic tests to identify and treat diseases have becomecommon tools in hospitals, homes and physician's offices. Biologicalfluids such as blood, urine or cerebrospinal fluids, which may at timescontain blood, are the most frequently employed biological samples forsuch tests.

Blood contains many different components, some of which are present instrikingly varied concentrations from sample to sample. The percentagesof both red and white blood cells in whole blood, for example, can varyamong normal individuals, and even in the same individual over time, andin particular under pathological conditions. This large variationcoupled with other factors such as storage conditions, coagulation, andthe fragility of red blood cells, produces considerable technicalproblems in performing diagnostics using blood-containing samples.

Whole blood is usually separated into various fractions prior totesting. Among other things, separation into fractions canadvantageously compensate for differences in hematocrit values, and inother ways reduce potential interference in up stream or down streambiochemical assays. Frequently employed fractions are serum, plasma,white cells, red blood cells and platelets. The terms “plasma” and“serum” are used herein to mean any fluid derived from whole blood fromwhich a substantial portion of the cellular components has been removed.Plasma and serum are used herein interchangeably, because the presenceor absence of coagulants is not a critical factor.

Blood separation technologies can be conceptually grouped into threecategories—centrifugation, filtration, and solid-phase separation.

Centrifugation

Blood separation is routinely achieved by centrifugation. Centrifugationis generally desirable because: (1) centrifugation can generallyseparate cellular components from serum or plasma at an efficiency ofgreater than 95%; (2) centrifuges do not require highly trainedpersonnel to operate; and (3) centrifugation allows concurrentprocessing of multiple samples in under 15 minutes. Centrifugation ofblood is, however, also problematic. For example, centrifuges areexpensive, involve multiple steps, are often unavailable at points ofcare such as bedside, schools or at home, and usually require electricalpower for operation.

Filtration

Many filtration techniques are known for separating various componentsfrom blood. U.S. Pat. No. 4,987,085 to Allen et al., for example,describes a filtering system with descending pore size using acombination of glass fiber membranes and cellulose membranes. U.S. Pat.No. 4,753,776 to Hillman et al. discloses a glass microfiber filterusing capillary force to retard the flow of cells. U.S. Pat. No.4,256,693 to Kondo et al. discloses a multilayered chemical analysiselement with filter layers made from at least one component selectedfrom paper, nonwoven fabric, sheet-like filter material composed ofpowders or fibers such as man-made fibers or glass fibers. U.S. Pat.Nos. 3,663,374 and 4,246,693 disclose membrane filters for separatingplasma from whole blood and U.S. Pat. Nos. 3,092,465, 3,630,957,3,663,374, 4,246,693, 4,246,107, 2,330,410 disclose further filtrationsystems, some of which make use of small-pore membranes.

Known filtration techniques generally reduce the volume of bloodrequired to only a few drops. Many filtration tests thereforecontemplate using only about 25 to 75 μl of whole blood. Some filtrationtechniques have even been developed that require only about 5 to 50 μlof whole blood. In most applications, filtration occurs directly on atest-strip in which the filtration surface is placed above the reactionzone or zones of the strip. Filtration in these formats also reduces oreliminates the availability problems associated with centrifuges.

But these advances often create entirely new problems. For example,filters tend to retain significant amounts of plasma, and analytespresent in low concentrations are frequently difficult to detect in theserum derived from small volumes of blood. Existing filters also tend toclog, and have undesirably slow flow rates. Agglutinating agents areoften mixed with whole blood to reduce clogging and to improve flowrates, (see U.S. Pat. Nos. 5,262,067, 5,766,552, 5,660,798 and5,652,148), but these problems remain.

Efforts have been made to improve the flow rate by modifying the forceemployed against the filter. But choices here are fairly limited.Filters are relatively simple to produce and use, but tend to causeexcessive hemolysis of red blood cells. Capillary action, a phenomenonin which water or liquid will rise above normal liquid level as a resultof attraction of molecules in liquid for each other and for the walls ofa capillary can also be used. Capillary action, however, is generallytoo weak to effect rapid separation of large volumes. (See, for example,U.S. Pat. Nos. 5,660,798, 5,652,148 and 5,262,067). Moreover, separationof plasma by capillary action tends to retain a relatively large amountof fluid within the wicking membrane, or a collection membrane. This inturn may necessitate testing the wicking membrane or the collectionmembrane or both, or eluting the retained material from the membranes.

Solid-Phase Separation

Solid-phase separation typically involves a surface having binding to atarget, the surface acting to immobilize and remove the target from asample. Exemplary solid-phase separation techniques are bindingchromatography, binding separation using beads, and hollow fibersseparations.

One particularly advantageous type of solid-phase separation is magneticseparation, in which a target is captured by magnetically attractable(paramagnetic) beads. Since no physical barriers are present, as wouldbe the case with filtration separation, magnetic separation tends to berelatively gentle. In U.S. Pat. No. 5,514,340 to Lansdorp and U.S. Pat.No. 5,123,901 to Carew, for example, magnetic wires are employed inbatch processes to separate magnetic particles from a fluid. In U.S.Pat. No. 4,663,029 to Kelland et al. and U.S. Pat. No. 5,795,470 toWang, magnetic particles are separated out from a fluid in a continuousflow process. Still other methods published for example in U.S. Pat. No.5,536,475 to Moubayed, employ rocking separation chambers and multiplemagnets to separate magnetic particles from a fluid.

One of the major limitations of applying known magnetic separation toblood separation is that multiple anti-ligands are required to removeall of the various types of cells and sub-cellular particles. Red bloodcells, lymphocytes, monocytes, and platelets, for example, havedifferent surface antigens, and do not specifically bind to any oneantibody. Furthermore, lack or absence of ligands on the cells due topathological conditions, genetic diseases or genetic variations or lifecycle of cells generally reduce the efficiency with which theanti-ligands bind with the target cells.

The problems with known magnetic separation devices are exacerbated withincreasing sample volumes, especially sample volumes over onemilliliter. Since many diagnostic applications require serum volumes ofup to one milliliter to satisfy the requirements of multiple tests orbatteries of tests, magnetic separation has not been particularlyuseful. Moreover, assays such as glucose or hemoglobin tests are highlysusceptible to interference caused by biological or chemical substancesin the sample, including proteins, bilirubin, and drugs.

Thus, there is still a need to provide improved methods and apparatusfor separating blood into its constituent parts, and especially forseparating plasma or serum from whole blood.

SUMMARY OF THE INVENTION

In accordance with the present invention, a cell-containing sample isseparated into a cell-containing portion and a substantiallycell-depleted portion, by mixing the sample with both an additive andparticles to produce a cell-containing network, and separating thenetwork from the remaining substantially cell-depleted portion using amagnetic force.

In one aspect of preferred embodiments the vessel has a plurality ofconfining walls, and at least one of the confining walls is flexible.The sample is retained within the confining walls, preferably compriseswhole blood, and the cell-containing portion largely comprises a networkof inter-linked red blood cells, stem cells, leukocytes, or lymphocytes.Especially preferred linkers include anti-ligands such as primaryantibodies that bind to a ligand or an antigen on or in the cellmembranes of the cells to be isolated/separated, and secondaryantibodies that bind to the primary antibodies. In another aspect ofpreferred embodiments, the primary antibodies are added directly to thesample, and the secondary antibodies are coupled to the surfaces ofparamagnetic beads.

In another aspect of preferred embodiments, polymeric materials such asPolybrene®, cationic liposomes, cationic lipids, and polydendromers maybe used in combination with anti-ligand(s) and magnetic separation or incombination with anti-ligand(s) and filtration. Aptamers can be used asanti-ligand(s) by themselves or in combination with cationic polymers,cationic liposomes, and dendromers.

In yet another aspect of preferred embodiments, the separation takesplace within the confining walls, and while in some embodiments theseparation employs at least in part a magnetic force, in otherembodiments at least two forces are employed to separate the networkfrom the substantially cell-depleted portion. Where two forces areemployed for separation, one force is a magnetic force and another forceis an electro-mechanical force transmitted through at least oneconfining wall, wherein the terms “electro-mechanical”, “automatic”,“hydraulic” and “pneumatic” are used interchangeably herein.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawings in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of a preferred embodiment in which separatingagents are being added to a blood sample.

FIG. 1B is a schematic of the embodiment of FIG. 1A following separationof red blood cells.

FIG. 2A is a schematic of an alternative embodiment.

FIG. 2B is a schematic of binding interactions contemplated to bepresent in the embodiment of FIG. 2A.

FIG. 3 is a schematic of another alternative embodiment.

FIG. 4 is a plan view of an exemplary diagnostic container.

FIG. 5 is a perspective view of an exemplary analyzer for a diagnosticcontainer of FIG. 4.

FIG. 6 is a photograph showing a detail view of an exemplary analyzerwith an actuator comprising a light guide for illumination and readingof scattered light.

FIG. 7A is a graph depicting a standard curve of measured hematocritagainst a reflectance signal obtained using an analyzer according to theinventive subject matter.

FIG. 7B is a graph depicting a correlation between spun hematocrit andhematocrit determined using a reflectance signal in an analyzeraccording to the inventive subject matter.

FIG. 8A is a graph depicting PSA measurements using various experimentalparameters.

FIG. 8B is a graph depicting a correlation between PSA measurement fromwhole blood using a reflectance signal obtained using an analyzeraccording to the inventive subject matter and PSA measurement frompreviously prepared plasma.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In the generalized preferred embodiment of FIG. 1A, a blood separationapparatus 10 comprises a vessel 20 and a magnet 50. The vessel 20contains blood 30, to which is being added primary antibodies 42 havinga substantial binding to cells, and particles 40 coated with a secondaryantibody having a substantial binding to primary antibodies 42.

Vessel 20 is preferably an ordinary test tube or test tube-like vesselsuch as a vacutainer or falcon tube. The volume of such tubes ispreferably less than about 10 ml, although it is contemplated thatappropriate vessels may define sample cavities of greater or lesservolumes.

Although vessel 20 is depicted as having a typical test-tube shape,alternative vessels are contemplated to have different shapes. Thus,suitable vessels may have a narrowed top portion to facilitate recoveryof the substantially red blood cell-depleted portion. Alternativevessels may also have differently shaped bottoms, such as a V-shaped ora flat bottom. In yet another example, an individual vessel may beformed as part of an array, such as in a multi-well microtiter plate.Further examples of suitable vessels include, hollow fibers, arrays ofcapillaries, beakers, pouches, dishes and cylinders—generally any devicethat can retain fluid within confining walls, and provide at least oneopening. Appropriate vessels are even contemplated to include “openwalled” structures such as a microscope slide having microchannelsetched on glass or plastic, or a simple plastic foil or film.

It is specifically contemplated that vessels employed in conjunctionwith the teachings herein retain a sample within a plurality ofconfining walls, and that at least one of the confining walls isflexible. The term “flexible” confining wall as used herein refers to awall that can be substantially deformed employing moderate pressure(i.e., less than 0.5 psi) without breaking or otherwise damaging theconfining wall. For example, a plastic foil or thin latex sheet areconsidered flexible. In contrast, a typical thin (2 mm) polycarbonateplate would not be considered flexible under the scope of thisdefinition, because polycarbonate plates can typically not besubstantially deformed without breaking Particularly contemplatedvessels may therefore have one relatively rigid wall onto which at leastone other confining flexible wall with individual compartments ismounted (e.g., a thin plastic sheet by heat sealing), and in even moreparticularly contemplated vessels, all of the confining walls may beflexible. Such vessels may have envelope shape and examples ofcontemplated vessels are described in U.S. patent application Ser. No.09/272,234, issued as U.S. Pat. No. 6,300,138, which is incorporatedherein by reference.

It is contemplated that vessels can be made from any appropriatematerial or materials, including glass, synthetic polymers, ceramics,metals, or mixtures thereof. Such vessel can be colored or transparent,translucent or non-translucent, and may or may not have graduation orother markings.

In FIG. 1A the sample being separated is whole blood. Such blood isgenerally contemplated to be fresh human whole blood, a few millilitersof which are preferably obtained by venipuncture. Another example iscapillary blood, which can be obtained in volumes ranging from less than10 to hundreds of μLs by use of a lancet. Furthermore, it iscontemplated that sample volumes higher than a few microliters to a fewmilliliters can be used, especially where a relatively high number ofcells is desired. For example, where lymphocytes are separated for usein a post-radiation transfusion in a cancer patient, suitable samplevolumes may be substantially higher than a few milliliters. Similarly,where stem cells for organ regeneration are separated, the sample volumewill likely exceed 50 ml and more. Therefore, contemplated volumes willtypically be in the range of about 1-10 mL, more typically in the rangeof 10-100 mL, and most typically in the range of about 100-500 mL (andeven more).

The blood can be pre-treated, such as by addition of an additive, orremoval of a component. Contemplated additives include buffer, water orisotonic solution, anticoagulants, antibodies, and test solutions.Contemplated substances or components that can be removed includeantibodies, globulins, albumin, and cellular fractions such asplatelets, white blood cells etc.

The blood can also be derived from non-human sources, includingvertebrate or invertebrate animals. Blood employed as set forth hereincan also be taken from any type of storage, and as such may be cooledblood, frozen blood, or blood with preservatives.

In preferred embodiments, the primary antibodies 42 are mouse derivedmonoclonal antibodies to human red blood cells, which in the field wouldoften be referred to as monoclonal Ab to hRBCs. The secondary antibodies44 are preferably sheep, donkey, goat, or mouse derived anti-mouse IgGantibodies.

Techniques for raising the antibodies are well known. For example, boththe primary and secondary antibodies can be derived from any appropriatesource including, goat, sheep, horse or recombinant sources. Suitableantibodies can also be selected from many classes and subclasses,including IgG and IgM, and subclasses. Furthermore, antibodies can beselected from numerous molecular varieties, including proteolyticfragments or engineered fragments such as Fab or (Fab)₂, or chimericantibodies. Combinations of antibodies are specifically contemplated.

Of course, both primary and secondary antibodies would advantageouslyhave substantial binding to their respective targets. The primaryantibodies would preferably have substantial binding to red blood cells,and in particular would have substantial binding to at least one ligandor component present on a surface of the red blood cells. The secondaryantibodies would preferably have substantial binding to at least somecomponent of the primary antibodies.

The secondary antibodies 44 are preferably included in the coating ofcoated particles 40. Such particles are attractable by magnetic force,and preferably comprise a paramagnetic composition embedded in syntheticpolymers or cellulose. Although paramagnetic particles are preferred,the coated particles can also or alternatively include ferromagnetic orchromium material or mixtures thereof. In still further variations,suitable particles can be coated with many other materials includingnatural or synthetic polymers, agarose etc. The preferred particle sizeis in the range of 0.1-100 μm, but alternative sizes between 10-100 nmor larger than 100 μm are also contemplated. Viewed from another aspect,it is contemplated to employ particles having a mean volume betweenabout 5×10⁻²⁴ m³ and about 5×10⁻⁶ m³. Where red blood cells are beingtargeted, the diameter of the red cells may advantageously be about fivetimes the diameter of the coated particles.

The term “coated” is used herein to mean any complete or partialcovering of any exposed surface. In FIG. 1, the particles 40 are coatedwith a material that immobilizes the secondary antibodies. Suchimmobilization can be temporary or permanent, and can involve covalentor non-covalent binding. For example, non-covalent binding may involveincubating antibodies with the bead or other solid-phase. As anotherexample, covalent coupling of antibodies to a solid-phase may involveincluding reacting amino groups of an antibody with aldehydes on thesolid-phase, or activated carboxyl groups on the solid-phase, resultingin a covalent bond.

In yet other embodiments, one or both of the primary and secondaryantibodies can be replaced by or complimented with an alternativecomposition have the desired binding, and at least a minimallyacceptable specificity. Anti-ligands are a general class of suchalternative compositions, and are defined herein as any molecule thatbinds non-covalently to an appropriate ligand. Examples of anti-ligandsand ligands include and are not limited to antibodies and antigens,respectively, and sense and anti-sense oligonucleotides in nucleicacids. Other polymers are contemplated as well as nucleotides.Additional examples are aptamers and lectins having a substantialbinding to ligands.

The term “a cell-containing network” refers herein to an aggregate of atleast more than one cell, from which individual cells cannot readily bemechanically removed without lysing the removed cells. Normally clottedblood is one example of a cell-containing network, but aggregates formedsubstantially by any combination mediated by molecular interactions suchas hydrophobic-, hydrophilic-, electrostatic-, van-der-Waals-, ionicinteraction or other molecular interactions are also contemplated. Thus,other examples of cell-containing networks are aggregates of red, whiteor other cells formed by combinations with antibodies or other linkingagents having substantial binding to the cells. It is especiallycontemplated that such networks may include solid supports such asbeads.

It is especially contemplated that heterogeneous aggregates can beformed using a mixture of red blood cells with two different antibodies,wherein the primary antibody binds the red blood cells, and thesecondary antibody binds to the primary antibody. If only one of the twobinding portions of the antibody is involved in such binding, thefollowing aggregates can be formed: (a) primary antibody bound to a redblood cell; (b) secondary antibody bound to the a primary antibody only;and (c) secondary antibody bound to primary antibody that is bound to ared blood cell or some other cell type in blood or body fluids. If bothof the two binding portions of the antibodies are involved in binding,any combination of the aggregates (a), (b), (c) may be formed, therebyproducing a potentially vast network of aggregates.

The sample, antibodies or other anti-ligands, and beads or otherparticles may be combined in the vessel in any order. For example, inone class of embodiments (not shown), vessels are contemplated to bepre-loaded with magnetically attractable beads. Suitable such vesselsare commercially available as MiniMACS™ separation columns from MiltenyiBiotec™, and the columns are even provided with a separation enhancingdevice. The standard protocol would need to be modified to conform tothe teachings herein, such as by pre-coating the beads with anappropriate anti-ligand, and by adding an appropriate anti-ligand to thesample.

The magnet 50 is generally a disc magnet, but in alternative embodimentsthe magnet can also have different shapes and designs. Contemplatedalternative magnets include bar magnets, horseshoe magnets, ringmagnets, and can have any suitable multiple pole geometry includingquadrapoles, hexapoles and octapoles, etc. Magnets can be of thepermanent type, electromagnets, or even super-conducting magnets, andmay comprise ferromagnetic or rare earth magnets. Furthermore, themagnet need not be a single magnet, but can advantageously comprise aplurality of magnets. Preferred magnets have strengths in the range of 0to 2 Tesla for permanent magnets, or 0-100 Tesla for electromagnets.Especially preferred magnets employ a permanent magnet of field strength0 to 1 Tesla.

In further alternative aspects of the inventive subject matter, thesample is retained within a plurality of confining walls, and theseparation takes place within the plurality of confining walls employinga first and a second force. While the first force comprises a magneticforce (vide supra), the second force comprises an automatic mechanicalforce that is transmitted through at least one of the confining walls.As used herein, the term “automatic” mechanical force is used herein torefer to a process in which a mechanical force is applied in a mannerother than manually applying a force. In particularly contemplatedembodiments, the automatic mechanical force comprises a pressure that isapplied to a flexible confining wall. For example, in a vessel whereinall of the confining walls are flexible, a magnetic force may retain thenetwork within the confining walls, while a mechanical force (e.g., oneor more actuators compressing the vessel) assist in separation of thenetwork from the substantially cell-depleted portion.

In FIG. 1B several networks 45 have been formed from the blood cells 32(not shown in detail), antibody coated paramagnetic particles 40 (notshown in detail), and anti-red blood cell antibodies 42 (not shown indetail). The particles 40 within the network 45 are being attracted bymagnet 50, thereby separating out the cell-containing networks 45 fromthe substantially cell-depleted plasma 34.

Red blood cells 32 are generally mature non-nucleated erythrocytes.These blood cells usually are the predominant form of red blood cellspresent in a sample. In alternative embodiments, red blood cells canalso be red blood cells carrying any type of hemoglobin including α, β,γ or fetal hemoglobin. The red blood cells can also be regular healthyblood cells or red blood cells giving raise to diseases e.g. sickle cellanemia or thalassemia. Appropriate red blood cells can also be in manystages of development, e.g. nucleated erythroblasts or aged,non-nucleated erythrocytes.

The inventive subject matter is, of course, not limited to maturenon-nucleated erythrocytes, and specifically contemplates other stagesof red cell development, other cells including white cells, and evencellular fragments including platelets. Thus, for example, where thesample comprises urine, a clinician or other individual may employ theinventive methods and apparatus to separate out bacterial cells orsloughed off bladder or urethral cells, and in such instances the redcells 32 of FIG. 1B may be replaced with non-erythrocytes.

Alternatively, various non-red blood cells may of particular interest,especially where such cells are separated for further clinical,experimental, or diagnostic use. Among particularly contemplatedalternative non-red blood cells, various stem cells, lymphocytes, andleukocytes are especially preferred. The term “stem cell” as used hereinrefers to a cell that gives rise to a lineage of cells, and that may becharacterized as a cell that upon division, produces dissimilardaughters, one replacing the original stem cell, the otherdifferentiating further. While it is generally preferred thatcontemplated stem cells are undifferentiated stem cells, partiallydifferentiated stem cells are also contemplated. Thus, suitable stemcells include embryonic stem cells, umbilical cord blood stem cells, andadult/peripheral stem cells. Consequently, the source of contemplatedsamples for use herein may vary considerably and may include blood,tissue samples (e.g., skin), commercially available stem cells and stemcell lines, and embryonic tissue. It is further contemplated that stemcells isolated by contemplated methods may be useful in a variety ofclinical and research settings, and particularly preferred uses includeat least partial regeneration of diseased or necrotic organs or organsubstructures, research into differentiation signals and pathways, etc.

Where isolated non-red blood cells are leukocytes, all subtypes of whiteblood cells are generally contemplated suitable and include neutrophiles(polymorphs), lymphocytes, eosinophiles, and basophiles. Of particularinterest are lymphocytes (i.e., white cells of the blood that arederived from stem cells of the lymphoid series), and especially variousB cells, T cells, T helper cells, and memory B cells. Such cells may beobtained from numerous sources, however, it is generally contemplatedthat the most common source are blood, amniotic fluid, and bone marrowaspirate. Thus, it is contemplated that leukocytes or lymphocytes may beobtained from any appropriate source (typically, the sample is takenfrom a mammal, and more preferably, from a human).

It is further contemplated that all markers that can both (i) form aspecific interaction for a desired cell type and (ii) allow a specificinteraction with a high affinity binding partner can generally be usedfor separation of the desired cell type. As used herein, the term “highaffinity binding partner” refers to an affinity of K_(d)<10⁻⁴ mol⁻¹.Consequently, numerous markers other than red blood cell markers (e.g.,glycophorin A) are suitable for use herein, and an exemplary collectionof suitable markers for various stem cells, leukocytes, and lymphocytesfor use in separation is provided in Table 1, below.

Marker Synonyms Specificity Leukocyte Markers (general) CD 1 Thymocytes,Langerhans histiocytes CD 33 Myeloid progenitor and monocytes S100Interdigitating dendritic cells of the lymph node paracortex. CD 45 LCA,leukocyte All leukocytes common antigen CD 30 Ki-I Activation marker forB, T, and monocytes T-Cell markers CD 2 T and NK cells CD 3 Allthymocytes, T and NK cells CD 4 Helper T cells CD 5 All T cells, some Bcells CD 7 All T cells, some myeloid cells CD 8 Cytotoxic T cells CD 16NK cells and granulocytes B-Cell markers CD 10 CALLA Early precursor andpre-B cells Common acute lymphocytic leukemia antigen CD 19 preB, Bcells, but not plasma cells CD 20 L26 preB, but not plasma cells CD 21EBV-R Mature B and follicular dendritic cells CD 22 Mature B CD 23Activated marrow B Other specific and general markers CD 15 Leu M2 Allgranulocytes, Reed Sternberg cells CD 34 Early pluripotent progenitorcell CD 61 platelet glycophorin Associated with M7 AML EMA epithelialmarker antigen Epithelial cells TdT T and B lymphocytes, lost beforematurity Stem Cell Markers SSEA-3 Primate embryonic stem cells SSEA-4Primate embryonic stem cells TRA-1-60 Primate embryonic stem cellsTRA-1-81 Primate embryonic stem cells Sca-1/Thy-1.1 lo Hematopoieticstem cells Hematopoietic stem cells

Where a particular antibody or affinity reagent for a marker for adesired stem cell, leukocyte, or lymphocyte is not commerciallyavailable, it is contemplated that suitable antibodies or antibodyfragments may be raised using standard procedures well known in the art(see e.g., Monoclonal Antibody Protocols (Methods in Molecular Biology,45) by William C. Davis; Humana Press; ISBN: 0896033082).

It should still further be appreciated that cells bound to the highaffinity binding partner (e.g., antibody or antibody fragment) can bereleased from such binding partners using a variety of techniques wellknown in the art, and particularly contemplated methods of releaseinclude competition with excess of antigen or epitope to which the highaffinity binding partner is specific, high temperature treatment,acidification (e.g., pH<3.5), proteolysis, and mild chaotropic agents.Where it is desirable that an isolated cell is removed from the magneticbead without separation of the high affinity binding partner from thecell, various methods of breaking a covalent bond between a highaffinity binding partner and a magnetic particle are contemplated. Amongsuch methods, reductive cleavage of a disulfide bond, or enzymaticcleavage (e.g., via an peptidase) of a bond in a linker connecting thehigh affinity binding partner to the magnetic particle are particularlypreferred. Exemplary methods of removing a hapten from an antibody aredescribed in Affinity Chromatography: Methods and Protocols (Methods inMolecular Biology) by Pascal Bailon, George K. Ehrlich, Wen-Jian Fung,wo Berthold, Wolfgang Berthold (Humana Press; ISBN: 0896036944), or inHandbook of Affinity Chromatography by Toni Kline (Marcel Dekker; ISBN:0824789393).

In addition to operating on a wide variety of samples, it is alsocontemplated that the inventive methods and apparatus described hereincan be employed to measure a wide variety of analytes. Contemplatedanalytes include tumor markers such as prostate specific antigen (PSA),infectious disease markers, endocrine markers such as testosterone,estrogen, progesterone and various cytokines, and metabolic markers suchas creatinine, glucose.

In FIG. 2A a blood separation apparatus 110 has a soft-walled orotherwise flexible vessel 120 containing a network 145 formed from wholeblood 130 comprising red blood cells 132 (not shown in detail), anti-redblood cell antibodies 142 (not shown in detail) and anti-mouseantibodies 144 (not shown in detail). A filter 160 filters out thenetwork 145, and allows plasma 134 to pass through to a collection area.

The filter 160 is preferably a glass fiber filter having a pore sizebelow the size of the cellular components of blood or larger than theindividual cells, but smaller than the network. In alternativeembodiments the filter can be made from many materials includingchromatographic paper, natural or synthetic fibers, porous membranesetc. Examples for those alternative filters are nylon fiber filters,size exclusion membranes, paper filters, woven fabric filters.Furthermore, the filters may or may not be coated with material e.g. toreduce hemolysis or to specifically retain selected fractions ormolecules. Appropriate coatings include polyvinylalcohol,polyvinylacetate, polycationic polymers, lectins or antibodies.

In FIG. 2A the filtrate portion of the sample is passed through thefilter by gravity. However, it is recognized that the driving force tomove the sample through the filter can be a force or pressuredifferential across the membrane, and can be achieved in many waysincluding centrifugation, vacuum, compressed gas, or a magnet asdescribed elsewhere herein. The filtration time can vary greatly, but isgenerally considered to be within the range of a few seconds to lessthan 30 min. Of course it should be appreciated that such separationcharacteristics and efficiency may also be obtained from various cells,other than red blood cells, including for example, leukocytes,lymphocytes, and stem cells.

FIG. 2B depicts details of a possible portion of network 145, whichincludes anti-red blood cell antibodies 142 bound to red blood cells132, and anti-mouse antibodies 144 bound to anti-red blood cellantibodies 142. Those skilled in the art will recognize that a singlenetwork can contain millions of cells, and it should be appreciated thatthe orientation and connections of the various components in FIG. 2B arepurely exemplary, and would not necessarily ever be found in an actualnetwork. Among other things, a real-life network would bethree-dimensional, rather than the two dimensional schematic as shown,and the antibodies would be much smaller than that shown in the drawing.It should also be appreciated that network 45 of FIG. 1B is contemplatedto have corresponding structures to that depicted in FIG. 2B.

In FIG. 3 a blood separation apparatus 210 has a vessel 220 thatreceives a blood sample 230, a pre-filter 260 coated with anti-red bloodcell antibodies 264, and a secondary filter 270 coated with anti-redblood cell antibodies 274. A portion of the sample 230 has filteredthrough the pre-filter 260 to provide a partially cell-depleted fluid233, and a portion of the cell-depleted fluid 233 has filtered throughthe secondary filter 270 to provide a substantially cell-depleted fluid234.

Here, the vessel 220 is contemplated to be a vessel falling withinbounds of vessels previously described with respect to vessel 20, andsimilar correspondences exist with respect to blood 230 and 30, redblood cells 232 and 32, primary antibodies 264 and 42, secondaryantibodies 274 and 44, and plasma 234 and 34.

The preferred pre-filter material is nylon wool 260, comprising anuncompressed layer of nylon fibers. The secondary filter 270 ispreferably a glass fiber disk onto which mouse anti-red blood cellantibodies 274 are bound. In alternative embodiments, either or both ofthe filters 260 and 270 can be substituted with any other suitablefilter material including a fibrous filter material, filter paper,porous membranes etc. Examples hereof include coated or uncoated glassfibers, mineral wool, chromatographic paper etc. Furthermore, the filtermaterial may or may not be coated e.g. to reduce hemolysis or tospecifically retain selected fractions or molecules. Appropriatecoatings include polyvinylalcohol, polyvinylacetate, polycationicpolymers, lectins or antibodies other than that previously described.

In FIG. 4, a plan view of an alternative and particularly preferredexemplary disposable diagnostic container 10 is shown and generallycomprises a pouch having a sample inlet port 12, a plurality ofcompartments 13, 22, 26, 28, 30, and 32, as well as passageway 16coupling the inlet port 12 with compartment 13, and portals 24, 34, 36,38 and 40 interconnecting the various compartments.

Container 10 is a relatively flat, laminated plastic pouch measuringabout 8.5 cm by about 19 cm, and about one millimeter thick, in whichthe compartments, inlet port, passageway and portals are all defined byheat sealing. The nature and dimensions of the container, arrangement ofcompartments and interconnections, as well as the contents of thecompartments will, of course, vary from embodiment to embodiment, andthose skilled in the art will recognize that the embodiment of FIG. 4 ismerely exemplary of an enormous number of such possible containers.

The size of the container, for example, largely depends on the volume ofreactants to be contained, although it is contemplated that practicalcontainers will typically be sized to define a volume in the range ofbetween 50 microliters and about 5 milliliters. Suitable containers mayhave many different shapes, so long as the shape permits contact of atleast one side of the container with a plurality of actuators. Preferredshapes are flat, envelope-like shapes, but box-like, round,hemispherical, or even spherical shapes, are also contemplated.

The opposing top and bottom sheets forming container 10 mayadvantageously be formed from a thermoplastic material, includingpolypropylene, polyester, polyethylene, polyvinyl chloride, polyvinylchloride, and polyurethane. Such sheets are contemplated to have arelatively uniform thickness between about 0.05 mm to about 2 mm. Theopposing sheets need not be fabricated from the same materials. Forexample, one sheet may comprise a reflective foil, and the other sheetmay comprise a transparent or translucent plastic. The use of foil canhelp promote temperature stability, and can serve as an additionalmoisture and oxygen barrier. Foil can also enhance thermal transfer froma heating source to a sample or reagent.

Preferred containers are flexible, either in whole or in part.Flexibility as characterized herein is the capability of yielding to areasonable force by temporarily changing shape without damaging thestructure or material. A reasonable force, as used herein, is apressure, typically below 5 lb/in². For example, a preferred flat,envelope-like container is sufficiently flexible to be wrapped around aninch diameter cylindrical object without breaking or tearing thecontainer. In another example, a portion of a container mayadvantageously be sufficiently flexible to displace a volume carriedwithin that portion without rupturing the outer walls. Moreover, it iscontemplated that at least part of the top and/or bottom sheets fromwhich the container may be fabricated is transparent or translucent. Thecontainer may furthermore have a plurality of openings. The number ofopenings may vary considerably between at least one opening and twentyopenings or more. Such openings may have a closing mechanism, besealable or permanently open. Furthermore, some of the openings may bein liquid communication with each other, or may be used as a vent or anoverflow. The container is furthermore characterized by having aplurality of compartments.

Container 10 also includes attachment holes 42 for mounting on alignmentposts in an analyzer 400. Alternative attachment devices or methods arealso contemplated, including hooks, loops and other mounting attachmentscoupled to the container 10 at appropriate locations. It is furthercontemplated that container 10 may be devoid of mounting components.

One or more labels (not shown) may also be affixed to the container 10.Labels may indicate identification marks, information relating to thetype of diagnostic test being conducted, as well as patient information,test result data, or other information. The label(s) may optionally beremovable, and may, for example, be removed from the container 10 to beplaced in a patient's medical file, thereby eliminating the need fortransferring data with attendant possibility for error.

Inlet port 12 serves as an entry point for receiving samples or othermaterials. Many configurations are contemplated, although it ispreferable that the entry point uses some sort of common connectionmechanism. For example, the entry point 12 in FIG. 4 is a female portionof a Luer lock mechanism. Alternative entry ports may be either simpleror more complex, and may contain a padding that can be punctured orpierced using a needle. Contemplated entry points may also be placedelsewhere on a container other than as depicted in FIG. 4. For example,a suitable entry point for a solid material may be formed as a simpleslot in one of the sheets forming the top or bottom of the container.Such an entry point may be well suited for receiving a relatively solidpiece such as a tissue or mineral sample, and may be sealable by a flapor tape mechanism.

Compartments 13, 22, 26, 28, 30, and 32 are portions of container 10that are fluidly separated from other portions of the container duringat least some period of time. In general, compartments are separatedfrom one another using at least one continuous element that contacts atleast one of the walls of the container. For example, if the containeris a cylinder, the continuous element could be a divider that is more orless perpendicular to the longitudinal axis of the cylinder, andcontacts the inner circumference of the cylinder. Where the container isa flat bag, the continuous element may advantageously comprise a heatseal between opposing sides, in a form enclosing a defined space.

The volume of preferred compartments may advantageously vary betweenabout 3% to approximately 90% of the total volume of the container. Suchcompartments may be filled with at sample, a reagent, or air, but thecompartment may also have essentially no void volume. By way of example,compartment 22 may be designed to contain about 1 ml of a bindingreactant, and wash compartment 28 may be designed to hold up to about 5ml of a solvent solution.

At least some of the compartments may advantageously comprise atransparent portion through which a signal can be detected, or theprogress of a reaction can be monitored. In such instances it may alsobe advantageous for an opposing surface to exhibit a reflective surfaceto improve signal detection. Compartments may also be shielded, forexample against heat, light, or other radiation. Especially preferredsignals that can be detected include signals from a luminescent and/orfluorescent marker, and scattered light (e.g., light scattered by cellsor cellular debris).

Compartments may have one or more openings, such as those at portals 34,36, 38, and 40. Such openings may be in permanent liquid communicationwith the rest of the container, for example, by an incomplete wallsurrounding the compartment. Openings may also be temporarily closed.For example, a breakable seal may form the opening, which separates thecompartment from the rest of the container, until an opening forcebreaks the seal. Typically, the breakable seal is a chevron break pointallowing a fluid to pass under about 5-15 psi. In another example, theopening comprises a one-way valve, which permits only a unidirectionalflow of material when a pressure difference is applied between the endsof the valve. In yet a further example, the opening may be temporarilyclosed by a closing force. Typically, the closing force is delivered viaa compression pad from outside the container, which effects a temporaryphysical separation of the compartment from the rest of the container.

Passageway 16 and portals 34, 36, 38 and 40 serve to fluidly connectvarious compartments and other spaces within the container, and with theexternal environment. The term “fluidly connect” specifically includesmovement of any fluidizable composition, whether a liquid, gas, orfluidized solid. In many instances the fluid will be intended to move ina single direction only, but in other instances it may be advantageousto move at least a portion of a fluid in both forward and backwardsdirections. In some cases compartments or other spaces may be separatedby a barrier for a period of time, and it is contemplated that thebarrier will at some point be breached. In such instances the separatedcompartments or other spaces are considered to be “fluidly connectable.”

Where containers according to FIG. 4 are employed, it is especiallypreferred that such containers cooperate with an automated analyzer asschematically depicted in FIG. 5. Here, an analyzer 400 generallycomprises a housing 410 having a container actuator assembly 412, a door420, a detector 440, a scatter unit 450, and an interface 460. Analyzer400 is shown with an exemplary work piece container 200. The housing 410houses essentially all of the electronic or other circuitry needed tocomplete the contemplated tests. Of course, housing 410 can be designedusing any suitable shape and dimensions, and can be formed from plastic,metal, or any other suitable materials. A container receiving zone withactuator assembly 412 cooperates with door 420 to receive container 10during the contemplated testing. In alternative embodiments a door isnot needed at all, and the container can instead be inserted into anaccess slot. Alignment posts 414 may be configured in any suitablefashion, and can be eliminated altogether.

Actuator assembly 412 is used to deliver one or more forces to thecontainer 10, with the object of affecting some material with container10. Examples of actuators that may form part of group 412 arecompression pads, roll bars, or wheels. Contemplated actuators may alsohave one or more additional functions, including heating, cooling,illuminating, and delivering a magnetic force. For example, an actuatormay heat inactivate an enzyme, or warm a reaction to a desiredtemperature. In another example, an actuator may be used to concentratean analyte by binding it the surface of a magnetic bead. Actuators mayalso be employed to modify a volume occupied by fluids, solids, or air.The fluids may, for example, include a buffer, a sample, a reactionmixture, a reagent solution, etc. The solids may include paramagneticbeads, and the gases may include nitrogen or argon as protective agents,or CO₂ as a byproduct of a chemical reaction.

Where an actuator comprises a compression pad, the pad can be made fromany material suitable for exerting an appropriate force to a portion ofa container, in an appropriate pattern. Typically, a compression pad isa substantially flat surface, and has a shape corresponding to the shapeof a compartment or passageway. Where an actuator is employed tootherwise seal a partition, a partitioning edge can be provided,preferably in the form of a wedge or a compression pad having aprotrusion.

Furthermore, in an alternative aspect of the inventive subject matter,the actuator group may include one actuator (which may act ascompression pad, heating pad, or serve other function) that includes alight source and/or a light detector (preferably, but not necessarily adetector other than the detector that detects an analyte signal). Forexample, it is contemplated that the actuator may include a laser diode(e.g., with wavelength of about 632 nm) and that the detector includes aphotocell that is spaced apart from the laser diode such that thedetector can detect light emitted by the laser diode that is scatteredfrom the cells in a cell-containing sample.

Of course, it should be recognized that the location of the light sourceand/or the light detector need not be limited to a compression pad, andin a more preferred aspect, the light source and/or the light detectormay be disposed in separate compression pads, or on opposite sides ofthe flat container, and in an even more preferred aspect, at least oneof the light source and/or the light detector are located in theanalytical device on the side opposite to the actuator group (i.e., theside or portion of the device that contacts the container, and that doesnot include the actuator group (e.g., the door, or adjacent to thesignal detector)). For example, where a device comprises a light sourceand a light detector on the same side (relative to the container), thedistance between the light source and the light detector is preferablybetween about 1 mm and 10 mm, and most typically about 3.5 mm.Alternatively, the function of the light detector may also be providedby the photomultiplier tube that is employed for detection of theanalyte signal, and the distance between the light source and thephotomultiplier may vary accordingly (e.g., typically between 1 mm and10 mm).

Moreover, it should be appreciated that the light source and/or thelight detector may be replaced with a light guide (e.g., fiber opticcable, or other light transmitting structure), such that one end of thelight guide is in optical communication with the cell-containing sample,and the other end of the light guide is in optical communication withthe light source or the light detector. An exemplary detail view of twolight guides an exemplary analyzer is shown in FIG. 6, wherein theanalyzer 600 (only part shown in detail view) has a scatter unit 610that includes a first light guide 620 (optically coupled to a lightsource) and a second light guide 630 (optically coupled to a photocell).Scatter unit 610 is proximal to a cavity that houses a photomultipliertube.

In still further alternative aspects, it is contemplated that the lightsource and wavelength may vary considerably. For example, suitable lightsources include numerous monochromatic and polychromatic light sources,and may provide incandescent, fluorescent, or laser light. Furthermore,where a particular wavelength is desired, one or more filters may beoptically coupled to the light source and/or the light detector. In yetfurther alternative aspects, the nature of the light detector may varyconsiderably and the same considerations as described below for thedetector 440 apply. Moreover, the inventors contemplate that the lightdetector may also be replaced by the detector 440 in FIG. 5.

Detector 440 is essentially one, or any combination of signal detectorsused to detect a signal generated through use of the container.Contemplated signal detectors include a photomultiplier tube, aphotodiode, and a charge-coupled device. It is optional to includedetector 440 in analyzer 400. An optional printer 450 is used to printinformation on any combination of human or machine-readable formats,including printing on a paper label or sheet. It is optional to includea printer in analyzer 400. Interface 460 can be any type of electronicor other means of exchanging information with another device. A typicalinterface is a common RS232 (serial) data port. Not shown are otheroptions for analyzer 400, including a scanner than can detect a barcode, or other hand or machine written information included on a label.

Depending on the wavelength of the light emitted by the light source, itshould be recognized that numerous measurement functions other thanlight scatter to determine hematocrit may be employed. For example, itis especially preferred that (after hematocrit is determined and acell-lysing agent is added to the blood sample), hemoglobin, and/oroxyhemoglobin may be spectrophotometrically determined in the samplecontainer using the appropriate wavelength. There are numerous methodsknown in the art to determine hemoglobin content and all of the knownmethods are considered suitable for use herein. For example, Bull et al.describe a standard photometric procedure in “Reference and SelectedProcedures for the Quantitative Determination of Hemoglobin in Blood;Approved Standard” (ISBN 1-56238-425-2), which is incorporated byreference herein.

It should be particularly appreciated that a combination of analyticalmethods using the light source and light detector (or photomultiplier)for a first analysis and the photomultiplier for a second analysis ofthe same sample may be especially advantageous where normalization ofthe second analysis result is desirable or indispensable. For example,glycosylated hemoglobin is frequently measured as a fraction ofglycosylated hemoglobin over total hemoglobin. Therefore, determinationof total hemoglobin in contemplated analytic devices may be performed ina spectrophotometric test using the light source and light detector ofthe scatter unit, while determination of glycosylated hemoglobin isperformed in an immunoassay using the photomultiplier. Of course, itshould be recognized that the particular nature of the first and secondtest is not limited to total hemoglobin and glycosylated hemoglobin. Infact, all known tests that require signal detection of two signals withdifferent wavelength and of different origin are contemplated suitablefor use herein. For example, the first signal may include an absorption,reflection, and/or scatter signal, while the second signal may be abioluminescence signal, a chemiluminescence signal, a fluorescencesignal, and/or a phosphorescence signal.

The analyzer can be programmable such that the compression pads andpartitioning edges apply particular external force at particular timesduring the diagnostic test. In addition, the analyzer apparatus can havean alignment means (e.g., a plurality of pins) for proper positioning ofthe container in the device. Further, the analyzer can have pressuresensors on either side of each compression pad and partitioning edge.These sensors can be used to determine and regulate the amount ofpressure being applied. In addition, these sensors can be used todetermine whether each compression pad and partitioning edge is workingproperly during operation. Similarly, the light source and the lightdetector may be operated at various times to provide an operator and/orsoftware light scatter information of a sample, and especiallycell-containing sample.

In general, a sample is deposited into inlet port 12 under pressure, andtravels to sample compartment 13. Excess sample beyond the capacity ofcompartment 13 spills over into a spillage compartment 20, which servesto aliquot the amount of sample in compartment 13. If the samplecomprises a cell-containing fluid (e.g., straight or diluted wholeblood, optionally admixed with one or more reagents), it is especiallypreferred that the light source emits a light into the sample(preferably with light having a maximum at a wavelength of between 620nm-660 nm) thereby generating a scattered light, which is then detectedby the light detector.

As a person of ordinary skill in the art will readily appreciate, theintensity of the so created scattered light in a cell-containing sampleis dependent on the amount of hematocrit in the cell containing sample.The term “hematocrit” as used herein refers to the totality of cells andcellular debris in a blood sample and therefore includes erythrocytes,leukocytes, thrombocytes, etc. There are numerous methods known in theart to calculate the hematocrit value in a cell-containing sample, andespecially in blood, and exemplary references for such determinationsinclude U.S. Pat. No. 6,419,822 and U.S. Pat. No. 6,064,474, which areincorporated by reference herein.

A first reactant from compartment 22 is added to the sample, and afterappropriate incubation the sample is shunted to reaction compartment 26.Reaction chamber 26 may contain additional reactants, and still furthermore reactants can be added from substrate or other reactant compartment30. At one or more points in the processing stage the sample can bewashed by a wash fluid from wash compartment 28. Waste material isforced into waste compartment 32. During these processes, variousreactions take place with respect to an analyte within the sample, and acolor or other detectable signal is produced that corresponds to theamount or existence of analyte. The signal is “read” through one of theside walls of compartment 26.

Where desirable, the read signal is then computed with the measuredscattered light to normalize the read signal to a value that correspondsto a value of the sample from which the hematocrit has previously beenremoved. Therefore, it should be particularly appreciated that usingcontemplated configurations and methods, an analyte can be detected in acell-containing sample (and preferably whole blood) without removing thecells from the sample.

EXPERIMENTS

Tests to separate red blood cells from plasma were performed, and theresults are described below. Further tests were performed to determinelevels of various antigens in cell-containing fluid without priorremoval of cellular components from the fluid. These tests are onlyintended to be illustrative of some of the principles set forth above,and are not intended to be read as limitations on the scope of theclaimed subject matter.

Experiment Set 1

In a first series of experiments, precipitation of red blood cells wasperformed using mouse anti-red blood cell antibodies, paramagnetic beadscoated with goat anti-mouse antibodies and 0.5 ml of anti-coagulatedwhole blood. Heparinized, EDTA or citrated whole blood was mixed with 10μl undiluted mouse anti-red blood cell antibody solution (Red Out™;Robbins Scientific Corp.) for 2 minutes; next 10 μl of a solution(isotonic PBS, pH 7.4) containing paramagnetic beads (Cortex BiochemInc., MagaCell™ or MagaBeads™; 30 mg/mL; or Pierce MagnaBind™) coatedwith goat anti-mouse antibodies was added and mixed gently for 2minutes. The bottom of the tube was placed on a magnet (permanent ironmagnet; approximately 0.2 Tesla). Precipitation of the cellular networkstarted instantly, and was substantially finished after about 2 minutes.Plasma was collected by aspiration from the top of the vessel.

Significantly, methods and apparatus described herein have been found toseparate at least 70% (by volume) of the theoretically availablecell-depleted portion from the network within a relatively short periodof time. In many cases the time period for such 90% separation is lessthan 30 minutes, in other cases less than ten minutes, and in stillother cases less than 2 minutes. Separations have also been performedusing the methods described herein that achieve at least 80%, at least90%, at least 95% and at least 98% of the theoretically availablecell-depleted portion from the network within less than 30 minutes.

It should especially be appreciated that such separation characteristicsand separation efficiencies may also be obtained using various cellsother than red blood cells, and especially contemplated alternativecells include various stem cells, lymphocytes, and leukocytes.

Experiment Set 2

In a second series of experiments, precipitation of red blood cells on amicroscope slide was performed using whole blood, mouse anti-red bloodcell antibodies and paramagnetic beads coated with goat anti-mouseantibodies. To 0.2 ml fresh whole blood, 5:l of an anti-red blood cellantibody containing solution (Red Out™) was added, mixed and incubatedfor 2 min at room temperature. Then, 5 :L of a solution containingparamagnetic beads coated with goat anti-mouse antibodies (MagaCell™ orMagaBeads™ or MagaBind™) was added, mixed, and after another 2 minutes,two disk magnets were positioned at opposite ends of the slide. Afterabout 1.5 minutes, a clear plasma containing zone was formed between thetwo magnets and this was retrieved with a pipette without disturbing thelaterally-fixed cell-containing network.

Experiment Set 3

Flat envelopes sealed on three of four sides were prepared fromtransparency acetate sheets (for example, the 3M Inc. product,3MCG3460), or plastic sheet protectors (e.g., Avery-Dennison PV119E), orfrom small “ZipLock” or ITW Inc. MiniGrip™ bags (2.5×3 cm; 2.0 mil).One-half to 1 mL of anti-coagulated whole blood was injected into thebag; 10 uL of Red Out™ (see above) was added and mixed with blood bygently rocking the container. After 2 minutes, the anti-mouse coatedmagnetic beads (MagaCell™) were added, mixed and the open side of thebag sealed. Two minutes later the bag was placed horizontally on arectangular permanent iron magnet (approximately 0.2 Tesla). Themagnetic particles and attached cellular networked moved adjacent to themagnet, leaving a clear layer of plasma as supernatant. The bag was thenrotated into an upright position while still on the magnet, opened, andplasma aspirated using a pipette. It was also discovered that envelopesor bags (containing blood previously treated with anti-RBC antibodiesand anti-mouse coated paramagnetic beads) could be passed between twopermanent magnets separated a narrow distance. As the sack was drawnupward between magnets, the cellular network was pulled to the bottom ofthe container, producing an overlying layer of plasma.

Thus, in this example a vessel retains a sample within a plurality offlexible confining walls, and a cell-containing network formed withinthe sample is separated within the plurality of confining walls.Alternatively, instead of passing the envelopes or bags between twomagnets, compression of the bag utilizing actuators or other mechanicaldevices could be employed to move the cellular network relative to thevessel. In this case, a second force (i.e., an automatic mechanicalforce) is employed to separate the network from the substantiallycell-depleted portion, wherein the second force is transmitted throughat least one of the confining walls.

Experiment Set 4

In one experiment, a small amount of steel wool (washed and treated with5 mg/mL BSA in PBS for 12 hours) was added to the sample container priorto addition of blood and precipitating reagents. After addition ofRedOut™ and MagaCell™ reagents (each for 2 minutes), the tube was placedon the magnet. The paramagnetic beads contained within the cellularnetwork were immobilized to the steel wool. A pipette tip was used foraspiration of plasma that was essentially free of blood cells. Whencoated with protein or some other polymer, the steel wool caused verylittle hemolysis in the cellular pellet in a 2-hr period. It is furthercontemplated that iron wire, wool, or beads could be added as above ifit were coated with other non-hemolyzing polymers such as dextran,polyvinylpyrilidone on polyethylene glycol etc.

In another experiment, 15 iron brads were taped around the externalbottom ⅓ of a 12 mm glass tube in which whole blood was incubated asdescribed above with mouse anti-red blood cell antibodies andparamagnetic beads coated with goat anti-mouse antibodies. Placing thetube on a rectangular magnet produced an almost immediate deposition onthe bottom and sides of the tube. The external brads place the magneticsource closer to the sample tube, thus applying a relatively uniformsource of secondary lateral attraction to the entire sample column andas the cellular network moves to the sides of the tube, it aggregatesfurther.

Experiment Set 5

Test results produced in accordance with methods and apparatus describedherein are depicted in Table 1. In this regard it should be noted thatcentrifugation is at least 99% effective in removing cellular matterfrom whole blood, (99% separation efficiency) and that methods andapparatus described herein (listed as “Device” in the table) are almostas effective. In particular, methods and apparatus described herein canbe described as having separation efficiency of least 90%, at least 95%,and at least 98%.

TABLE 1 Plasma Testosterone Creatinine Volume mL PSA Ng/mL ng/mL mg/dLCentri- Centri- Centri- Centri- Subject fugation Device fugation Devicefugation Device fugation Device 1 0.45 0.36 28.5 27.2 4.78 5.45 0.750.64 2 0.55 0.48 25.1 23.2 14.45 11.2 0.70 0.62 3 0.53 0.48 27.2 26.416.19 15.18 1.04 1.06 4 0.48 0.40 26.9 24.8 11.18 10.96 0.97 0.91 5 0.570.51 25.9 24.2 0.84 0.91 6 21.3 20.2 10.97 10.15 1.11 1.10 7 25 2 23.66.08 7.58 0.69 0.72 8 25.5 20.4 18.06 22.79 0.99 0.91 9 16.4 16.6 0.460.27 1.35 1.49 Red White Hemoglobin Plasma Blood Cells Blood Cells g/dLHematocrit % millions/uL thousands/uL Centri- Centri- Centri- Centri-Subject fugation Device fugation Device fugation Device fugation Device1 0.27 0.16 2 0.27 0.19 3 0.38 0.24 4 0.25 0.15 5 0.48 0.13 6 0.12 0.05<0.1 <0.1 0 0 0.2 0.1 7 0.16 0.08 <0.1 <0.1 0 0 0.2 0.2 8 0.22 0.09 <0.1<0.1 0 0.02 0.2 0.2 9 0.33 0.17 <0.1 <0.1 0 0 0.2 0.2

Experiment Set 6

An analyzer as depicted in FIG. 5 was employed for measurement ofreflectance of whole blood in dependence of the hematocritconcentration. In this particular experiment, analyzer 400 had a housing410 and actuator assembly 412. Holding pins 414 retained container 200(see also FIG. 4) in a predetermined position relative to the actuatorassembly 412, photomultiplier 442, and scatter unit 450. The scatterunit 450 included a first optical fiber port 452 that retained anoptical fiber, wherein the fiber was anchored in the fiber port 452 suchthat one end of the fiber contacted the container (having onetransparent side) and illuminated whole blood disposed within thecontainer, while the other end of the optical fiber was illuminated by alaser diode with a wavelength maximum of 632 nm.

The scatter unit 450 further included a second optical fiber port 454that retained another optical fiber, wherein the fiber was anchored inthe fiber port 454 such that one end of the fiber contacted thecontainer (having one transparent side) and received light scattered bythe cells in the whole blood, while the other end of the optical fiberwas optically coupled to a photodiode. The signal of the photodiode waselectronically amplified and recorded using standard equipment wellknown in the art.

To determine a correlation between hematocrit concentration andreflectance in such configurations, whole blood samples with knownhematocrit concentrations were individually placed into the containerand illuminated while the container was disposed in the analyzer. Theresults of this experiment are depicted in FIG. 7A, in which reflectancewas found to be a linear function of hematocrit with a high correlationcoefficient.

Experiment Set 7

Using the analyzer of the previous experiment, whole blood samples withknown hematocrit concentrations were divided in two portions. Oneportion was spun and the hematocrit concentration determined inconventional manner. The other portion was placed into the container andhematocrit was determined as described above using the linear functionof FIG. 7A. FIG. 7B depicts the correlation between the hematocritmeasurement as determined by reflectance and the hematocrit measurementas determined by centrifugation. Again, a relatively high correlationcoefficient confirmed that hematocrit can be accurately determined usingreflectance in the analyzer of Experiment 6.

Experiment Set 8

Using the analyzer of the previous experiment, free PSA (prostatespecific antigen) was determined from whole blood using monoclonalantibodies whole blood samples with known hematocrit (here: betweenabout 15-60%) and free PSA (here: between 8-10 ng/ml) concentrations. Ina first set of experiments, the whole blood samples were employed formeasurement without prior centrifugation to remove cellular components.As expected and can be seen from FIG. 8A, the actual PSA signaldecreased with increasing hematocrit concentrations. In contrast, wherethe blood samples were centrifuged to remove cellular components, thePSA concentration remained at the expected levels. Similarly, where theblood samples were not centrifuged, but normalized using the correlationestablished from the experiments 6 and 7 above, the PSA concentrationremained at substantially the same level, and showed high correlation tothe centrifuged blood samples.

Of course, it should be recognized that the analytes that can bedetected using methods and configurations according to the inventivesubject matter need not be restricted to free PSA. It is generallycontemplated that all analytes (and especially analytes that aredissolved or disposed in whole blood) for which a colorimetric,luminometric, or fluorimetric assay is available are consideredsuitable. For example, suitable analytes will include peptides (e.g.,viral antigens, growth factors, CEA, etc.), hormones (e.g., TSH ortestosterone), nucleic acids (e.g., tumor associated nucleic acids),small molecules (e.g., Schedule I-IV drugs, T4/T4 thyroxin, etc.).

Experiment Set 9

Using the analyzer and correlations determined in the previousexperiments, free PSA was determined over a range of PSA concentrationsin samples over a range of hematocrit concentrations. Whole blood wasused as starting material for two sets of experiments. In a first set ofexperiments, free PSA was determined from whole blood without removal ofthe cellular components using reflectance and correction factorsobtained from the previously determined correlations. In a second set ofexperiments, free PSA was determined from whole blood after removal ofcellular components. As can be clearly seen in FIG. 8B, there is anexcellent correlation between the two experiments. Consequently, itshould be appreciated that free PSA can be determined from whole bloodin contemplated configurations without prior removal of cellularcomponents, which will allow a physician to perform a free PSA test inhis of her office without a requirement of an otherwise need permit toprocess (e.g., centrifuge) whole blood.

Consequently, the inventors contemplate a method of testing acell-containing sample for a analyte in which in one step a container isprovided wherein the container has a plurality of fluidly discontinuouscompartments, including a sample reacting compartment, different firstand second reagents contained in first and second reagent compartments,respectively, and a signal detection compartment. In another step, acell-containing sample is placed into the sample reacting compartment,and the cell-containing sample is illuminated in the container therebycreating a light scatter signal (a light scatter signal is acquired). Ina still further step, a surface of the container is contacted with adevice having a plurality of actuators, and multiple different sets ofthe actuators are operated to independently add the first and secondreagents to the sample in a variable sequence and time delay. In yetanother step, at least a portion of the reacted sample is moved betweenthe sample reacting compartment and the sample detection compartmentusing at least one of the sets of actuators, and an analyte-dependentsignal is read from the reacted sample contained in the sample detectioncompartment, and a test result is calculated using the analyte-dependentsignal and the light scatter signal.

Viewed from another perspective, the inventors therefore contemplate amethod of manipulating a container in which a container is providedhaving a reaction channel fluidly coupled to a plurality ofcompartments, including a sample receiving compartment and a signaldetection compartment, wherein at least one of the compartments includesa reagent. In another step, a cell-containing sample is introduced intothe sample receiving compartment, and the cell-containing sample isilluminated in the container thereby creating a light scatter signal,which is acquired. In yet another step, a first portion of a surface ofthe container is contacted with a device having a plurality ofindependently operable actuators, wherein at least one of the actuatorscompresses a portion of the container thereby moving at least part ofthe sample into the reaction channel, and in yet another step, a secondportion of the surface of the container is contacted with another one ofthe actuators that compresses a portion of the container therebypreventing a movement of at least one of the part of the sample and thereagent between at least two of the plurality of compartments. In stillanother step, an analyte-dependent signal is read from the reactedsample contained in the sample detection compartment, and a test resultis calculated using the analyte-dependent signal and the light scattersignal.

Thus, specific embodiments and applications of magnetic separation havebeen disclosed. It should be apparent to those skilled in the art,however, that many more modifications besides those already describedare possible without departing from the inventive concepts herein. Theinventive subject matter, therefore, is not to be restricted except inthe spirit of the appended claims.

1. A method of manipulating a container, comprising: providing aflexible container having a reaction channel fluidly coupled to aplurality of compartments, including a sample receiving port and asignal detection compartment, wherein at least one of the compartmentscontains a reagent; introducing a cell-containing sample into the samplereceiving port, and moving the sample from the sample receiving port toone of the plurality of compartments; illuminating the cell-containingsample via a transparent portion of the container in the one of theplurality of compartments of the container thereby creating a lightscatter signal, and reading the light scatter signal; contacting a firstportion of a surface of the container with a device having a pluralityof independently operable actuators, wherein at least one of theactuators compresses a portion of the container thereby moving at leastpart of the sample into the reaction channel; contacting a secondportion of the surface of the container with another one of theactuators that compresses a portion of the container thereby preventinga movement of at least one of the part of the sample and the reagentbetween at least two of the plurality of compartments; moving the partof the sample in at least one of a unidirectional and bi-directionalmovement between the sample receiving port and the signal detectioncompartment using the plurality of actuators; and using the lightscatter signal to calculate a hematocrit value based on a known functioncorrelating previously determined spun hematocrit values and lightscatter signals.
 2. The method of claim 1 further comprising providingthe device with a signal detector, and detecting an analyte signal usingthe signal detector.
 3. The method of claim 2 further comprisingcalculating a test result for the cell-containing sample using theanalyte signal and the light scatter signal.
 4. The method of claim 3wherein the step of calculating comprises correlating the light scattersignal with the calculated hematocrit value.
 5. The method of claim 1wherein the step of illuminating is performed using a light guidedisposed in one of the actuators.
 6. The method of claim 1 wherein thestep of reading the light scatter signal is performed using anotherlight guide disposed in the one of the actuators.
 7. The method of claim1 wherein the step of illuminating is performed using light having awavelength maximum at between 620 nm and 660 nm.
 8. The method of claim1 wherein the flexible container comprises a flexible top sheet and aflexible bottom sheet.